n.
Any of various techniques for the separation of complex mixtures that rely on the differential affinities of substances for a gas or liquid mobile medium and for a stationary adsorbing medium through which they pass, such as paper, gelatin, or magnesia.
chromatographer chro'ma·tog'ra·pher n.
chromatography
Dictionary:
chro·ma·tog·ra·phy (krō'mə-tŏg'rə-fē)  |
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A physical separation method in which the components of a mixture are separated by differences in their distribution between two phases, one of which is stationary (stationary phase) while the other (mobile phase) moves through it in a definite direction. The substances must interact with the stationary phase to be retained and separated by it.
Retention results from a combination of reversible physical interactions that can be characterized as adsorption at a surface, absorption in an immobilized solvent layer, and electrostatic interactions between ions. When the stationary phase is a porous medium, accessibility to its regions may be restricted and a separation can result from size differences between the sample components. More than one interaction may contribute simultaneously to a separation mechanism. The general requirements are that all interactions must be reversible, and that the two phases can be separated (two immiscible liquids, a gas and a solid, and so forth) in such a way that a distribution of sample components between phases and mass transport by one phase can be established. See also Absorption; Adsorption.
Reversibility of the interactions can be achieved by purely physical means, such as by a change in temperature or by competition; the latter condition is achieved by introducing substances into the mobile phase that have suitable properties to ensure reversibility for the interactions responsible for retention of the sample components. Since this competition with the sample components is itself selective, it provides a general approach to adjusting the outcome of a chromatographic experiment to obtain a desired separation. It is an absolute requirement that a difference in the distribution constants for the sample components in the chromatographic system exist for a separation to be possible.
Methods
A distinction between the principal chromatographic methods can be made in terms of the properties of the mobile phase and configuration of the stationary phase. In gas chromatography the mobile phase is an inert gas, in supercritical fluid chromatography the mobile phase is a fluid (dense gas above its critical pressure and temperature), and in liquid chromatography the mobile phase is a liquid of low viscosity. The stationary phase can be a porous, granular powder with a narrow particle-size distribution packed into a tube (called a column) as a dense homogeneous bed. This configuration is referred to as a packed column and is nearly always used in liquid chromatography and is commonly used in supercritical fluid and gas chromatography. Alternatively, the stationary phase can be distributed as a thin film or layer on the wall of an open tube of capillary dimensions, leaving an open space through the center of the column. This configuration is referred to as an open tubular column (or incorrectly as a capillary column); and it is commonly used in gas chromatography, frequently used in supercritical fluid chromatography, but rarely used in liquid chromatography.
Thin-layer chromatography is a form of liquid chromatography in which the stationary phase is spread as a thin layer over the surface of a glass or plastic supporting structure. The stationary phase must be immobilized on the support by using a binder to impart the desired mechanical strength and stability to the layer. The samples are applied to the layer as spots or bands near the bottom edge of the plate. The separation is achieved by contacting the bottom edge of the plate below the line of samples with the mobile phase, which proceeds to ascend the layer by capillary action. This process is called development and is performed in a chamber, with the lower edge of the layer in contact with the mobile phase and the remaining portion of the layer in contact with solvent vapors from the mobile phase. The chamber may be a simple device such as a covered jar or beaker or a more elaborate device providing control of the mobile-phase velocity and elimination or control of the vapor phase. Thin-layer chromatography is the most popular form of planar chromatography having virtually replaced paper chromatography in laboratory practice. See also Gas chromatography; Gel permeation chromatography; Liquid chromatography; Supercritical-fluid chromatography.
Uses
Chromatographic methods provide a means of analyzing samples (to determine component identity and relative amount), of isolating significant quantities of purified material for further experimentation or commerce, and for determining fundamental physical properties of either the samples or the mobile or stationary phases (for example, diffusion coefficients, solubilities, or thermodynamic properties). There are virtually no boundaries to the sample types that can be separated. Examples include organic and inorganic compounds in the form of fixed gases, ions, polymers, as well as other species. Applications are found in all areas of technological development, making chromatography one of the most widely used laboratory procedures in chemistry. Depending on intent, chromatography can be applied to trace quantities at the limit of detector response (for example, 10−15 g) or to kilogram amounts in preparative separations.
Instrumentation
Modern chromatographic methods are instrumental techniques in which the optimal conditions for the separation are set and varied by electromechanical devices external to the column or layer. Separations are largely automated, with important features of the instrumentation being control of the flow and composition of the mobile phase, introduction of the sample onto the stationary phase, and on-line detection of the separated components. In column chromatography the sample components are detected in the presence of the mobile phase after they have exited the stationary phase. In thin-layer chromatography the sample components are detected in the presence of the stationary phase, resulting in different detection strategies.
Instrument requirements differ by the needs of the method employed. Gas chromatography, for example, employs a mobile phase of constant composition at a few atmospheres of column inlet pressure and variation in the temperature of the column to effect a separation. Liquid chromatography uses a pump to select or vary the composition of the mobile phase with a high column inlet pressure (typically a few hundred atmospheres) and a constant temperature for the separation. These differences in optimized separation conditions result in different equipment configurations for each chromatographic method.
Interpretation
The results of a chromatographic experiment are summarized in a chromatogram (see illustration), a two-dimensional record of the detector response to the sample components (y axis) plotted against the residence time of the components in column chromatography or migration distance in planar chromatography (x axis). Individual compounds or mixtures of unseparated compounds appear as peaks in the chromatogram. These peaks are ideally symmetrical and occur at positions in the chromatogram that are characteristic of their identity, with a distribution around the mean position (apex of the peak) that is characteristic of the kinetic properties of the chromatographic system. The area inscribed by the peak is proportional to the amount of substance separated in the chromatographic system.
Typical chromatogram obtained by gas chromatography.
Information readily extracted from the chromatogram includes an indication of sample complexity (the number of observed peaks), qualitative substance identification (determined by peak position), relative composition of the sample (peak dimensions; area or height), and a summary of the kinetic characteristics of the chromatographic system (peak shapes).
| Dental Dictionary: chromatography |
n
Any one of several processes for separating and analyzing various gaseous or dissolved chemical materials according to differences in their absorbency with respect to a specific substance.
| Britannica Concise Encyclopedia: chromatography |
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A technique for separating and analysing the components of a mixture of liquids or gases. Chromatography depends on the selective absorption of the different components in a column of powder (column chromatography) or on a strip of paper (paper chromatography). Chromatography is one of the techniques used to identify specific drugs in a urine sample.
| Columbia Encyclopedia: chromatography |
chromatography (krō'mətŏg'rəfē), resolution of a chemical mixture into its component compounds by passing it through a system that retards each compound to a varying degree; a system capable of accomplishing this is called a chromatograph. The retarding system can be a surface adsorbant, such as silica, alumina, cellulose, or charcoal, capable of reversibly adsorbing the compounds (see adsorption). The earliest use of this technique, by the Russian botanist Mikhail Tsvett (c.1903), involved the separation of highly colored compounds, hence the name chromatography [Gr.,=color recording].
Column Chromatography
In column chromatography the adsorbant is packed into a column and a solution of the mixture is added at the top. An appropriate solvent is passed through the column, washing, or eluting, the compounds down the column. A polar substance that is adsorbed very tightly to the surface will be efficiently retarded by the column, while a nonpolar substance will elute (dissolve in the solvent) very rapidly. By varying the nature of the solid adsorbant and the eluting solvent, a wide variety of resolutions, even of very similar substances, can be carried out.
Gas Chromatography
The gas chromatograph (GC) is a system consisting of a liquid with a high boiling point impregnated on an inert solid support as the stationary phase and helium gas as the mobile phase. The stationary phase is packed into a thin metal column and helium gas is allowed to flow through it. The column is attached to an injection port, and the entire system is heated in an oven. A solution of the mixture is injected into the column through the injection port by means of a syringe and is immediately volatilized. The helium gas then sweeps the components out of the column and past a detector. The polarity of the compounds and their volatility determines how long they are retained by the column. When each component passes the detector, a peak is registered on a recorder. The relative quantities of the components can be determined from the relative areas under the peaks. By varying the polarity of the column and its temperature, many different resolutions can be carried out. Since the capacity of GC columns is very low, the gas chromatograph is used chiefly as an analytical tool, although it can be used for preparative purposes as well. Miniaturized GC instruments have been employed in space probes to analyze the atmospheres of other planets.
Liquid Chromatography
For compounds that cannot be volatilized readily, the liquid chromatograph (LC) can be used instead of the gas chromatograph. The stationary phase consists of a finely powdered solid adsorbant packed into a thin metal column and the mobile phase consists of an eluting solvent forced through the column by a high-pressure pump. The mixture to be analyzed is injected into the column and monitored by a detector. Many different LC packings and eluting solvents are available to achieve the desired resolution.
Gel-Permeation Chromatography
In gel-permeation chromatography, compounds are separated on the basis of their molecular size. Porous beads of the gel are packed into a column and the mixture is added at the top in an appropriate solvent. Large molecules move straight down the column, while small molecules stick in the pores and are retarded.
Ion-Exchange Chromatography
For compounds that can exist as ions, ion-exchange chromatography can be used to separate them from neutral or oppositely charged compounds. The mixture is added to a column packed with a porous, insoluble resin which has a negatively charged (anionic) group attached to it and an unattached, positively charged (cationic) counterion. A cation from the mixture will exchange with the positive counterion of the resin and will be retarded while neutral and anionic substances are not affected. Ion-exchange resins with exchangeable anions work in a similar manner.
Thin-Layer and Paper Chromatography
A layer of adsorbant also can be spread on a glass plate, instead of packed into a column, for analytical purposes. By means of a thin capillary tube, the plate is spotted with a solution of the mixture that is to be resolved, and the solvent is allowed to evaporate. An eluting solvent is then allowed to move up the plate by capillary action, drawing the components of the mixture along by varying degrees. The plate is developed by spraying it with an oxidizing agent, so that each component becomes charred and appears as a dark spot on the plate. The location and size of the spots serve to identify and measure the relative quantities of the components. As in column chromatography, polar substances will not elute as well and will remain nearer the bottom of the plate, while nonpolar substances will elute to the top. This process is called thin-layer chromatography (TLC). In paper chromatography a procedure similar to TLC is used except that the cellulose in the paper acts as the adsorbant.
Electrophoresis
Electrophoresis, like ion-exchange chromatography, can be used as an effective tool for analyzing mixtures of ions. A strip of paper or a column of polymeric gel, saturated with an electrolyte, is set up so that it spans two solutions containing electrodes. The mixture to be analyzed is spotted onto the paper or gel and the two electrodes are connected to a high-energy power source (about 5,000 volts). Positive ions will migrate in one direction and negative ions in the other. The greater the charge on the ion, the farther it will migrate. This method is especially useful for the resolution of mixtures of proteins.
| Veterinary Dictionary: chromatography |
A technique for analysis of chemical substances. The term chromatography literally means color writing, and denotes a method by which the substance to be analyzed is poured into a vertical glass tube containing an adsorbent, the various components of the substance moving through the adsorbent at different rates, according to their degree of attraction to it, and producing bands of color at different levels of the adsorption column. The term has been extended to include other methods utilizing the same principle, although no colors are produced in the column.
The mobile phase of chromatography refers to the fluid that carries the mixture of substances in the sample through the adsorptive material. The stationary phase (or adsorbent) refers to the solid material that takes up the particles of the substance passing through it. Kaolin, alumina, silica and activated charcoal have been used as adsorbing substances or stationary phases.
Classification of chromatographic techniques tends to be confusing because it may be based on the type of stationary phase, the nature of the adsorptive force, the nature of the mobile phase, or the method by which the mobile phase is introduced.
The technique is a valuable tool for the research biochemist and is readily adaptable to investigations conducted in the clinical laboratory. For example, chromatography is used to detect and identify in body fluids certain sugars and amino acids associated with inborn errors of metabolism.
- adsorption c. — that in which the stationary phase is an adsorbent.
- affinity c. — a method of chromatography that utilizes the biologically important binding interactions that occur on protein surfaces. For example, an enzyme substrate is covalently coupled to an inert matrix such as a polysaccharide bead. The enzyme can be bound to the bead and thereby separated when present in very low concentration in a very complex mixture of other macromolecules.
- column c. — the technique in which the various solutes of a solution are allowed to travel down a column, the individual components being adsorbed by the stationary phase. The most strongly adsorbed component will remain near the top of the column; the other components will pass to positions farther and farther down the column according to their affinity for the adsorbent. If the individual components are naturally colored, they will form a series of colored bands or zones. — Column chromatography has been employed to separate vitamins, steroids, hormones and alkaloids and to determine the amount of these substances in samples of body fluids.
- exclusion c. — that in which the stationary phase is a gel having a closely controlled pore size. Molecules are separated based on molecular size and shape, smaller molecules being temporarily retained in the pores.
- gas c. — a type of chromatography in which the mobile phase is an inert gas. Volatile components of the sample are separated in the column and measured by a detector. The method has been applied in the clinical laboratory to separate and quantify steroids, barbiturates and lipids.
- gas–liquid c. — gas chromatography in which the substances to be separated are moved by an inert gas along a tube filled with a finely divided inert solid coated with a nonvolatile substance; each component migrates at a rate determined by its solubility in the stationary phase and its vapor pressure.
- gel-filtration c., gel-permeation chromatography — exclusion chromatography.
- high performance liquid c. (HPLC) — a miniaturized method in which the solution to be analyzed is passed, under high pressure, through a long, thin column packed with tiny beads such that analyses are completed in minutes rather than hours and with improved resolution.
- ion-exchange c. — that utilizing resins to which are coupled either cations or anions that will exchange with other cations or anions in the material passed through their meshwork.
- molecular sieve c. — exclusion chromatography.
- paper c. — a form of chromatography in which a sheet of special paper is substituted for the adsorption column. After separation of the components as a consequence of their differential migratory velocities, they are stained to make the chromatogram visible. In the clinical laboratory paper chromatography is employed to detect and identify sugars and amino acids.
- partition c. — a form of separation of solutes utilizing the partition of the solutes between two liquid phases, namely the original solvent and the film of solvent on the adsorption column.
- thin-layer c. — that in which the stationary phase is a thin layer of an adsorbent such as silica gel coated on a flat plate. It is otherwise similar to paper chromatography.
| Wikipedia: Chromatography |
Chromatography (from Greek χρώμα:chroma, color and γραφειν:graphein to write) is the collective term for a set of laboratory techniques for the separation of mixtures. It involves passing a mixture dissolved in a "mobile phase" through a stationary phase, which separates the analyte to be measured from other molecules in the mixture based on differential partitioning between the mobile and stationary phases. Subtle differences in compounds partition coefficient results in differential retention on the stationary phase and thus changing the separation.
Chromatography may be preparative or analytical. The purpose of preparative chromatography is to separate the components of a mixture for further use (and is thus a form of purification). Analytical chromatography is done normally with smaller amounts of material and is for measuring the relative proportions of analytes in a mixture. The two are not mutually exclusive.
Contents |
History
Main article: History of chromatography
The history of chromatography begins during the mid-19th century. Chromatography, literally "color writing", was used—and named— in the first decade of the 20th century, primarily for the separation of plant pigments such as chlorophyll. New types of chromatography developed during the 1930s and 1940s made the technique useful for many types of separation process.
Some related techniques were developed during the 19th century (and even before), but the first true chromatography is usually attributed to Russian botanist Mikhail Semyonovich Tsvet, who used columns of calcium carbonate for separating plant pigments during the first decade of the 20th century during his research of chlorophyll.
Chromatography became developed substantially as a result of the work of Archer John Porter Martin and Richard Laurence Millington Synge during the 1940s and 1950s. They established the principles and basic techniques of partition chromatography, and their work encouraged the rapid development of several types of chromatography method: paper chromatography, gas chromatography, and what would become known as high performance liquid chromatography. Since then, the technology has advanced rapidly. Researchers found that the main principles of Tsvet's chromatography could be applied in many different ways, resulting in the different varieties of chromatography described below. Simultaneously, advances continually improved the technical performance of chromatography, allowing the separation of increasingly similar molecules.
Chromatography terms
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This article is in a list format that may be better presented using prose. You can help by converting this article to prose, if appropriate. Editing help is available. (September 2008) |
- The analyte is the substance that is to be separated during chromatography.
- Analytical chromatography is used to determine the existence and possibly also the concentration of analyte(s) in a sample.
- A bonded phase is a stationary phase that is covalently bonded to the support particles or to the inside wall of the column tubing.
- A chromatogram is the visual output of the chromatograph. In the case of an optimal separation, different peaks or patterns on the chromatogram correspond to different components of the separated mixture.
- Plotted on the x-axis is the retention time and plotted on the y-axis a signal (for example obtained by a spectrophotometer, mass spectrometer or a variety of other detectors) corresponding to the response created by the analytes exiting the system. In the case of an optimal system the signal is proportional to the concentration of the specific analyte separated.
- A chromatograph is equipment that enables a sophisticated separation e.g. gas chromatographic or liquid chromatographic separation.
- Chromatography is a physical method of separation in which the components to be separated are distributed between two phases, one of which is stationary (stationary phase) while the other (the mobile phase) moves in a definite direction.
- The effluent is the mobile phase leaving the column.
- An immobilized phase is a stationary phase which is immobilized on the support particles, or on the inner wall of the column tubing.
- The mobile phase is the phase which moves in a definite direction. It may be a liquid (LC and CEC), a gas (GC), or a supercritical fluid (supercritical-fluid chromatography, SFC). A better definition: The mobile phase consists of the sample being separated/analyzed and the solvent that moves the sample through the column. In one case of HPLC the solvent consists of a carbonate/bicarbonate solution and the sample is the anions being separated. The mobile phase moves through the chromatography column (the stationary phase) where the sample interacts with the stationary phase and is separated.
- Preparative chromatography is used to purify sufficient quantities of a substance for further use, rather than analysis.
- The retention time is the characteristic time it takes for a particular analyte to pass through the system (from the column inlet to the detector) under set conditions. See also: Kovat's retention index
- The sample is the matter analysed in chromatography. It may consist of a single component or it may be a mixture of components. When the sample is treated in the course of an analysis, the phase or the phases containing the analytes of interest is/are referred to as the sample whereas everything out of interest separated from the sample before or in the course of the analysis is referred to as waste.
- The solute refers to the sample components in partition chromatography.
- The solvent refers to any substance capable of solubilizing other substance, and especially the liquid mobile phase in LC.
- The stationary phase is the substance which is fixed in place for the chromatography procedure. Examples include the silica layer in Chromatography#Thin layer chromatography
Techniques by chromatographic bed shape
Column chromatography
For more details on this topic, see Column chromatography.
Column chromatography is a separation technique in which the stationary bed is within a tube. The particles of the solid stationary phase or the support coated with a liquid stationary phase may fill the whole inside volume of the tube (packed column) or be concentrated on or along the inside tube wall leaving an open, unrestricted path for the mobile phase in the middle part of the tube (open tubular column). Differences in rates of movement through the medium are calculated to different retention times of the sample.[1]
In 1978, W. C. Still introduced a modified version of column chromatography called flash column chromatography (flash).[2][3]The technique is very similar to the traditional column chromatography, except for that the solvent is driven through the column by applying positive pressure. This allowed most separations to be performed in less than 20 minutes, with improved separations compared to the old method. Modern flash chromatography systems are sold as pre-packed plastic cartridges, and the solvent is pumped through the cartridge. Systems may also be linked with detectors and fraction collectors providing automation. The introduction of gradient pumps resulted in quicker separations and less solvent usage.
A spreadsheet that assists in the successful development of flash columns has been developed. The spreadsheet estimates the retention volume and band volume of analytes, the fraction numbers expected to contain each analyte, and the resolution between adjacent peaks. This information allows users to select optimal parameters for preparative-scale separations before the flash column itself is attempted.[4]
In expanded bed adsorption, a fluidized bed is used, rather than a solid phase made by a packed bed. This allows omission of initial clearing steps such as centrifugation and filtration, for culture broths or slurries of broken cells.
Planar Chromatography
Planar chromatography is a separation technique in which the stationary phase is present as or on a plane. The plane can be a paper, serving as such or impregnated by a substance as the stationary bed (paper chromatography) or a layer of solid particles spread on a support such as a glass plate (thin layer chromatography). Different compounds in the sample mixture travel different distances according to how strongly they interact with the stationary phase as compared to the mobile phase. The specific Retardation factor (Rf) of each chemical can be used to aid in the identification of an unknown substance.
Paper Chromatography
For more details on this topic, see Paper chromatography.
Paper chromatography is a technique that involves placing a small dot or line of sample solution onto a strip of chromatography paper. The paper is placed in a jar containing a shallow layer of solvent and sealed. As the solvent rises through the paper, it meets the sample mixture which starts to travel up the paper with the solvent. This paper is made of cellulose, a polar substance, and the compounds within the mixture travel farther if they are non-polar. More polar substances bond with the cellulose paper more quickly, and therefore do not travel as far.
Thin layer chromatography
For more details on this topic, see Thin layer chromatography.
Thin layer chromatography (TLC) is a widely-employed laboratory technique and is similar to paper chromatography. However, instead of using a stationary phase of paper, it involves a stationary phase of a thin layer of adsorbent like silica gel, alumina, or cellulose on a flat, inert substrate. Compared to paper, it has the advantage of faster runs, better separations, and the choice between different adsorbents. For even better resolution and to allow for quantitation, high-performance TLC can be used.
Displacement Chromatography
The basic principle of displacement chromatography is: A molecule with a high affinity for the chromatography matrix (the displacer) will compete effectively for binding sites, and thus displace all molecules with lesser affinities.[5] There are distinct differences between displacement and elution chromatography. In elution mode, substances typically emerge from a column in narrow, Gaussian peaks. Wide separation of peaks, preferably to baseline, is desired in order to achieve maximum purification. The speed at which any component of a mixture travels down the column in elution mode depends on many factors. But for two substances to travel at different speeds, and thereby be resolved, there must be substantial differences in some interaction between the biomolecules and the chromatography matrix. Operating parameters are adjusted to maximize the effect of this difference. In many cases, baseline separation of the peaks can be achieved only with gradient elution and low column loadings. Thus, two drawbacks to elution mode chromatography, especially at the preparative scale, are operational complexity, due to gradient solvent pumping, and low throughput, due to low column loadings. Displacement chromatography has advantages over elution chromatography in that components are resolved into consecutive zones of pure substances rather than “peaks”. Because the process takes advantage of the nonlinearity of the isotherms, a larger column feed can be separated on a given column with the purified components recovered at significantly higher concentrations.
Techniques by physical state of mobile phase
Gas chromatography
For more details on this topic, see Gas chromatography.
Gas chromatography (GC), also sometimes known as Gas-Liquid chromatography, (GLC), is a separation technique in which the mobile phase is a gas. Gas chromatography is always carried out in a column, which is typically "packed" or "capillary" (see below) .
Gas chromatography (GC) is based on a partition equilibrium of analyte between a solid stationary phase (often a liquid silicone-based material) and a mobile gas (most often Helium). The stationary phase is adhered to the inside of a small-diameter glass tube (a capillary column) or a solid matrix inside a larger metal tube (a packed column). It is widely used in analytical chemistry; though the high temperatures used in GC make it unsuitable for high molecular weight biopolymers or proteins (heat will denature them), frequently encountered in biochemistry, it is well suited for use in the petrochemical, environmental monitoring, and industrial chemical fields. It is also used extensively in chemistry research.
Liquid chromatography
Liquid chromatography (LC) is a separation technique in which the mobile phase is a liquid. Liquid chromatography can be carried out either in a column or a plane. Present day liquid chromatography that generally utilizes very small packing particles and a relatively high pressure is referred to as high performance liquid chromatography (HPLC).
In the HPLC technique, the sample is forced through a column that is packed with irregularly or spherically shaped particles or a porous monolithic layer (stationary phase) by a liquid (mobile phase) at high pressure. HPLC is historically divided into two different sub-classes based on the polarity of the mobile and stationary phases. Technique in which the stationary phase is more polar than the mobile phase (e.g. toluene as the mobile phase, silica as the stationary phase) is called normal phase liquid chromatography (NPLC) and the opposite (e.g. water-methanol mixture as the mobile phase and C18 = octadecylsilyl as the stationary phase) is called reversed phase liquid chromatography (RPLC). Ironically the "normal phase" has fewer applications and RPLC is therefore used considerably more.
Specific techniques which come under this broad heading are listed below. It should also be noted that the following techniques can also be considered fast protein liquid chromatography if no pressure is used to drive the mobile phase through the stationary phase. See also Aqueous Normal Phase Chromatography.
Affinity chromatography
For more details on this topic, see Affinity chromatography.
Affinity chromatography[6] is based on selective non-covalent interaction between an analyte and specific molecules. It is very specific, but not very robust. It is often used in biochemistry in the purification of proteins bound to tags. These fusion proteins are labelled with compounds such as His-tags, biotin or
Supercritical fluid chromatography
For more details on this topic, see Supercritical fluid chromatography.
Supercritical fluid chromatography is a separation technique in which the mobile phase is a fluid above and relatively close to its critical temperature and pressure.
Techniques by separation mechanism
Ion exchange chromatography
For more details on this topic, see Ion exchange chromatography.
Ion exchange chromatography uses ion exchange mechanism to separate analytes. It is usually performed in columns but can also be useful in planar mode. Ion exchange chromatography uses a charged stationary phase to separate charged compounds including amino acids, peptides, and proteins. In conventional methods the stationary phase is an ion exchange resin that carries charged functional groups which interact with oppositely charged groups of the compound to be retained. Ion exchange chromatography is commonly used to purify proteins using FPLC.
Size exclusion chromatography
For more details on this topic, see Size exclusion chromatography.
Size exclusion chromatography (SEC) is also known as gel permeation chromatography (GPC) or gel filtration chromatography and separates molecules according to their size (or more accurately according to their hydrodynamic diameter or hydrodynamic volume). Smaller molecules are able to enter the pores of the media and, therefore, take longer to elute, whereas larger molecules are excluded from the pores and elute faster. It is generally a low resolution chromatography technique and thus it is often reserved for the final, "polishing" step of a purification. It is also useful for determining the tertiary structure and quaternary structure of purified proteins, especially since it can be carried out under native solution conditions.
Special techniques
Reversed-phase chromatography
For more details on this topic, see Reversed-phase chromatography.
Reversed-phase chromatography is an elution procedure used in liquid chromatography in which the mobile phase is significantly more polar than the stationary phase.
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This section requires expansion. |
Two-dimensional chromatography
In some cases, the chemistry within a given column can be insufficient to separate some analytes. It is possible to direct a series of unresolved peaks onto a second column with different physico-chemical (Chemical classification) properties. Since the mechanism of retention on this new solid support is different from the first dimensional separation, it can be possible to separate compounds that are indistinguishable by one-dimensional chromatography.
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This section requires expansion. |
Simulated Moving-Bed Chromatography
For more details on this topic, see Simulated moving bed.
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This section requires expansion. |
Pyrolysis gas chromatography
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This section requires expansion. |
Fast protein liquid chromatography
For more details on this topic, see Fast protein liquid chromatography.
Fast protein liquid chromatography (FPLC) is a term applied to several chromatography techniques which are used to purify proteins. Many of these techniques are identical to those carried out under high performance liquid chromatography, however use of FPLC techniques are typically for preparing large scale batches of a purified product.
Countercurrent chromatography
For more details on this topic, see Countercurrent chromatography.
Countercurrent chromatography (CCC) is a type of liquid-liquid chromatography, where both the stationary and mobile phases are liquids. It involves mixing a solution of liquids, allowing them to settle into layers and then separating the layers.
Chiral chromatography
Chiral chromatography involves the separation of stereoisomers. In the case of enantiomers, these have no chemical or physical differences apart from being three dimensional mirror images. Conventional chromatography or other separation processes are incapable of separating them. To enable chiral separations to take place, either the mobile phase or the stationary phase must themselves be made chiral, giving differing affinities between the analytes. Chiral chromatography HPLC columns (with a chiral stationary phase) in both normal and reversed phase are commercially available.
See also
References
- ^ IUPAC Nomenclature for Chromatography IUPAC Recommendations 1993, Pure & Appl. Chem., Vol. 65, No. 4, pp.819-872, 1993.
- ^ Still, W. C.; Kahn, M.; Mitra, A. J. Org. Chem. 1978, 43(14), 2923-2925. (doi:10.1021/jo00408a041)
- ^ Laurence M. Harwood, Christopher J. Moody. Experimental organic chemistry: Principles and Practice (Illustrated edition ed.). pp. 180-185.
- ^ Fair, J. D.; Kormos, C. M. J. Chromatogr. A 2008, 1211(1-2), 49-54. (doi:10.1016/j.chroma.2008.09.085)
- ^ Displacement Chromatography 101. [1] Sachem, Inc. Austin, TX 78737
- ^ Pascal Bailon, George K. Ehrlich, Wen-Jian Fung and Wolfgang Berthold, An Overview of Affinity Chromatography, Humana Press, 2000. ISBN 978-0-89603-694-9, ISBN 978-1-60327-261-2.
External links
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- Library 4 Science: Chrom-Ed Series
- Overlapping Peaks Program - Learning by Simulations
- Chromatography Videos - MIT OCW - Digital Lab Techniques Manual
- Chromatography Equations Calculators - MicroSolv Technology Corporation
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| Dental Dictionary. Mosby's Dental Dictionary. Copyright © 2004 by Elsevier, Inc. All rights reserved. Read more |
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| Britannica Concise Encyclopedia. Britannica Concise Encyclopedia. © 2006 Encyclopædia Britannica, Inc. All rights reserved. Read more |
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| Sports Science and Medicine. The Oxford Dictionary of Sports Science & Medicine. Copyright © Michael Kent 1998, 2006, 2007. All rights reserved. Read more |
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| Columbia Encyclopedia. The Columbia Electronic Encyclopedia, Sixth Edition Copyright © 2003, Columbia University Press. Licensed from Columbia University Press. All rights reserved. www.cc.columbia.edu/cu/cup/. Read more |
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| Veterinary Dictionary. Saunders Comprehensive Veterinary Dictionary 3rd Edition. Copyright © 2007 by D.C. Blood, V.P. Studdert and C.C. Gay, Elsevier. All rights reserved. Read more |
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| Wikipedia. This article is licensed under the Creative Commons Attribution/Share-Alike License. It uses material from the Wikipedia article "Chromatography". Read more |
Related answers
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